Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (α1H) T-type Ca2+ Channels

摘要

Voltage-gated T-type Ca²⁺ channel Cav3.2 (α1H) subunit, responsible for T-type Ca²⁺ current, is expressed in different tissues and participates in Ca²⁺ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Cav3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca²⁺ in different cells. We examined the localization and regulation of the Cav3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Cav3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Cav3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Cav3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Cav3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (ICa,T) and adenovirus (AdCav3.2)-mediated increase in peak Cav3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of ICa,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Cav3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca²⁺ signaling during normal and pathological processes.